The advent of novel and highly promising chemical and biochemical methods of analysis which have found application on an ever increasing scale in recent years in medicine and industry for research purposes calls for the provision of apparatus capable of taking very small measured volumes of several sample liquids to be dispensed into a single test tube. These methods include biochemical modelling and genetic engineering, immunology, enzymological and bioluminescent methods, as well as kinetic methods of clinical analysis. The methods are based on the preparation of a sample of a multicomponent mixture of solutions of high purity. To this end, very small measured volumes of sample liquids are dispensed into and simultaneously mixed in a single test tube. Therewith, the volumes of each of the measured sample solutions, depending on the specific aims of the analysis, vary within two samples, whereas the measured volumes of the solutions are dispensed and mixed either simultaneously, or by a controllable delay in dispensing of one or more components thereof. The latter affords not only to improve the accuracy and reproducibility of the analyses, but also to obtain added information on the character of the reactions constituting the method resulting in new data relating to the substances under analysis.
For example, in enzymological and bioluminescent methods, as well as in the methods of biochemical modelling and genetic engineering, determination of the kinetic parameters of the analytical reactions enables to evaluate not only the quantity of the biomolecules, but also the functional properties of the molecules and those of the activation centers thereof. On the other hand, in all of the above stated analytical methods each of the substances dispensed into the test tube features a concentration exceeding the value required for reaction to proceed, this being necessitated by the concentration to be reduced to a normal value in the course of adding other sample components. Rapid and simultaneous mixing of all sample components may, therefore, prove a decisive factor for a majority of analytical reactions to take place. This can be accounted for by the fact that many biologically active molecules subject to analysis tend to irrevocably alter their structure and lose the functional activity at high concentrations of a number of salts, although small concentrations of the latter are an essential factor for initiating the reaction.
A device for micrometering several sample liquids to be dispensed into a single test tube must therefore provide, in accordance with a programmable analysis, automatic variations in the volume and time of liquid sample dispensing along each of the channels, as well as high reproducibility and accuracy of dispensing.
When dispensing liquid samples measured in microliters, it is important that a provision be made for separating from the end of a sample liquid conveying tube a measure of liquid in volume by far less than a drop thereof. Incomplete separation of the sample liquid from the end of the conveying tube may reduce several-fold the accuracy of metering. Therefore, along with the provision of a high precision sample metering means, it is just as important to provide a liquid sample dispensing assembly that would meet the above requirement.
An advantage of the above methods resides in their high sensitivity, which to a large extent depends on the purity of reagents used for analysis. For example, the bioluminescent method enables to detect 10.sup.-13 of a gram of substance. Conversely, the presence in the sample under analysis of even negligeable quantities (in the order of tenths of one percent) of admixtures including the solutions used for analysis, reduces the sensitivity of the method by several orders of magnitude. This requirement to maintain the purity of the solutions relates in equal measure both to the measured samples and to the initial sample liquids, which imposes additional limitations on the construction of the sample conveying assembly.
As has been stated above, in the multiple-component reactions use is made of solutions with high concentration of substance therein. This is explained not only by the need to dilute the solution in the course of the preparation of a multicomponent mixture, but also by the necessity to obtain a highly reactive mixture. The latter applies mostly to the analysis of biomolecules. Therefore, it is almost always undesirable to dilute the sampled solution by adding water or flushing fluids used for flushing the pipes and metering vessels for changing the sample liquids.
The use in said methods of highly pure and costly reagents poses another requirement to devices for micrometering sample liquids, viz. the sample liquids must remain in the interior of pipes, syringes and containers after the sample liquid have been measured.
There is known a micrometering sampler (cf. USSR Inventor's Certificate No. 463,027, IPC G 01 N 1/10, G 01 F 11/06, published Mar. 5, 1975) comprising piston-and-cylinder syringes secured on a bracket with the piston rods thereof extending upwards to cooperate with a common driver. The bracket also mounts adjusting screws for presetting the volume of liquid being sampled.
With the pistons of the syringes in the downmost position, the bracket is manually lowered for the needles of the syringes to enter vessels containing sample liquids. The driver is then raised to draw into the syringes the sample liquids, whereafter the bracket is raised to replace the sample containers with empty test tubes to receive the thus sampled liquid. The bracket is then again lowered for the measured sample liquid to fill the test tubes by pressing the driver.
Insufficient automation in the above device determines the low efficiency of dispensing several sample liquids into a test tube. Another disadvantage of the apparatus resudes in a low accuracy of liquid metering because the syringes are positioned with their piston rods extending upwards, which impedes the removal of air from the interior thereof during drawing in the sample liquid.
Flushing the interior of the syringes subsequent to each dispensing operation reduces further the efficiency of the apparatus, while the flushing fluid remaining in the interior and the walls of the syringes and needles thereof result in dilution of the sampled liquid. In cases where dilution is inadmissible, the needles are not flushed which in turn leads to cross contamination of the sample liquid and measured samples thereof.
Also known is a micromeasuring liquid sampler (cf. U.S. Pat. No. 3,991,616, Cl. B 01 L 3/02, published Nov. 16, 1976) comprising a syringe with the piston rod thereof extending upwards and having several openings to hold pipes therein. Each of the pipes is made up of three sections fabricated respectively from polyamide, plasticized polyvinyl chloride and stainless steel, the polyamide sections being fixedly secured in the openings of the syringe. The plasticized polyvinyl chloride sections in conjunction with solenoid controlled pipe pinching means form a shut off unit of each of the pipes. The steel sections are immersed in liquid sample containing vessels. A container is provided for a flushing fluid, while a test tube is further provided for receiving measured quantities of the sample liquids. The piston rod is connected to a step-by-step drive to impart reciprocations thereto.
During the upward movement of the piston, all the pipes are closed except one intended to convey either any of the sample liquids or the flushing fluid. The sample liquid flows along this pipe into the interior of the syringe. During the downward movement of the piston all the pipes are closed except one intended for dispensing the sample liquid into the test tube. The flushing fluid is the last to be conveyed to wash the interior of the syringe.
A disadvantage of the above apparatus resides in that removal of air from the interior of the syringe is complicated due to the piston rods being positioned to extend upwards; such an arrangement of the piston rods results in more time to be consumed for making the apparatus ready to operate and leads to considerable analytical errors because of the air bubbles formed during the suction of viscous gas-containing liquids and remaining in the interior of the syringe.
Conveying the sample liquids in succession through one syringe makes it necessary to flush the syringe subsequent to each sample preparation, while in order to completely clean the interior of the syringe, the latter should be flushed at least 10 times, which is described in detail in Analytical Biochemistry, vol. 86, 1978, pp. 1-20 - Christian Stahly, John H. Wharton, Hans Noll "A Computer Controlled Multichannel Micropipetter".
On the one hand, this limits the efficiency of the micromeasuring liquid sampler, while on the other it becomes a source of cross contamination, since the experimentally found requirement to flush the sampler ten times is sometimes not sufficient to completely clean the sampler. The foregoing does not permit to obtain samples free from the flushing fluid, which in turn results in reduced concentration of the sample components and lower the reactivity thereof.
Also, for successive dispensing of all the solutions of the multicomponent sample by one syringe sampler, the need to take into consideration the volume of sample liquid in the pipe and make corrections due to free play in the connections of the drive for reciprocating the piston rod add to inconveniencies for the operator.
Further known is a liquid sampler comprising syringes equal in number to the number of sample liquids being measured and provided with pipes for conveying and dispensing the liquids into a test tube arranged on a tray, the syringes being secured in a stand kinematically linked with a sample volume regulator in the form of a drive for reciprocating the pistons of the syringes relative to the cylinders thereof, the drive being electrically connected to a sample volume and timing setter forming part of a control unit, the control unit being electrically wired with another drive means for imparting reciprocations to the ends of the sample conveying pipes (cf. brochure "Sample Processor" of the Kone Company, Finland).
The outlets of the syringes are connected by pipes to respective probes secured on a bracket linked with a drive for imparting a step-by-step vertical and horizontal movement thereto. The position of each probe is such that it corresponds to the position of a respective test tube of three rows of tubes equal in number to the number of the sample liquids. Arranged horizontally under the bracket in the path of travel thereof are the tube carrying tray, vessels containing the sample liquids being metered and a flushing fluid container having a means for mechanically cleaning the probes.
The apparatus is controlled by a microprocessor. Upon depressing respective buttons, the bracket is caused to move horizontally toward the flushing fluid container to be lowered in the downmost position, whereby the syringes, pipes and probes are filled with the flushing fluid by several reciprocations of the syringe pistons.
From their downmost position the pistons are moved upwards a distance corresponding to a preselected volume of the first sample liquid. Therewith, the bracket moves upwards and is displaced horizontally toward the first sample liquid to be thereafter lowered into the vessel containing the liquid. The pistons are then moved to their downmost position to draw the sample liquid, after which the bracket is again raised and moved toward the first row of test tubes and lowered to introduce the probes into the test tubes. The pistons are then moved upwards for the first sample liquid to fill the test tubes, whereupon the bracket is raised, moved toward the flushing fluid container and again lowered. The upward movement of the pistons causes a portion of the flushing fluid to be forced out of the probes. The bracket is then raised, which is accompanied by the probes passing through the mechanical probe cleaning means, whereafter the sampling and dispensing cycle is repeated for the second sample liquid etc., depending on the number of sample liquid employed.
However, the drive for reciprocating the piston rods of the syringes fails to permit variations in the amount of each of the sample liquids being drawn thereinto, which substantially restricts the range of application of the apparatus.
Due to all of the piston rods being driven by a common drive, it is impossible to vary the time of dispensing, which in turn impedes the use of the apparatus for conducting kinetic analyses requiring simultaneous dispensing and simultaneous mixing all the liquid sample components. For the same reason, the apparatus cannot be used for a number of analyses of the functionally active biomolecules wherein the effect of high salt concentration is inadmissible, although the salts are necessary for the reaction of the low concentration sample components. Another drawback resides in that the apparatus cannot be used for such analyses which require successive adding the components making up the sample, since the interval between successive dispensing into the sample of two liquids cannot be made less than one dispensing cycle exceeding in this apparatus ten seconds.
Further, some free play may be present in the movable connections between the drive and the piston in the course of reciprocations impairing the accuracy of sampling, the free play being determined by the structural arrangement of the drive for moving the pistons of the syringes, limiting the operating cycle by only one measure of sample liquid being taken.
Due to the volume of the sampled liquid being very small, the drop of liquid may not fall into the test tube, for which reason the end of the probe is introduced directly into the already measured and dispensed liquid. This results in that a portion of the liquid is likely to be carried by the end of the probe, the volume of the thus withdrawn liquid amounting to 30-40 microliters, which is practically equal to the volume of one liquid drop. This is inadmissible for most of the above analyses, because the final sample containing all the measured sample liquids may have the same volume. Therefore, the above apparatus features low accuracy and cannot be used for a number of analyses.
In spite of the provision for the flushing fluid and probe cleaning means, the sample liquids and the measured samples are subject to cross contamination. Further, it is the flushing fluid and the probe cleaning means that cause such a cross contamination. Each operating cycle is accompanied by at least some of measured sample liquid being carried by the probe from the test tube containing the mixture of measured sample into the flushing fluid. As a result, when metering highly concentrated solutions after only several cycles the concentration of the sample substances in the flushing fluid may reach tenths of one percent. Secondly, after each cycle of sample liquid dispensing a small amount of the sample liquid being measured is added to the flushing fluid by the probe, which also leads to a gradual build up therein of the substances diluted in the sample liquids. Thirdly, the mechanical probe cleaning means fails to provide cleaning of the entire surface area of the probe, which results in transfer of the flushing fluid to the measured samples along with all the substances present therein. Besides, the probe cleaning assembly requires accurate alignment of the probes relative thereto.
Filling the flush fluid wetted interiors of the probe and pipes with the sample liquid being measured dilutes the sample liquid during each measuring cycle resulting in less accurate sample liquid metering and errors in subsequent analyses. Dilution of the sample liquid components also brings down the rate of reaction which rules out kinetic analyses to be carried out.
Multiple flushing the outer surfaces of the probes and interior thereof as well as the interior of the sample liquid conveying pipes leads to losses of the sample liquids being measured, which is highly undesirable when analyzing very pure substances. Conversely, flushing is an obligatory operation, since the construction of the sample liquid metering means, the drive thereof and the reciprocating liquid dispensing probes necessitates sequential metering different sample liquids by each of the syringes, which in turn requires flushing respective pipes during each sample metering cycle. Also, the flushing fluid is liable to penetrate the sample liquid due to incomplete cleaning of the probes. Therefore, the remainder of the sample liquids in the containers are not suitable for further use after a series of analyses have been conducted, which likewise results in losses of the sample liquids.